The major histocompatibility complex of humans (denoted HLA-human leukocyte antigen) is a cluster of genes occupying a region located on the sixth chromosome. The polymorphic proteins encoded by the HLA region have been designated HLA-A,-B,-C,-DR,-DQ, and -DP. HLA-A, -B, and -C consist of a single polymorphic chain. HLA-DR, -DQ, and -DP proteins contain two polymorphic chains, designated alpha and beta. These D-region proteins are encoded by loci designated DRA, DRB1, DRB3, DRB4, DQA1, DQB1, DPA1, and DPB1.
The products encoded by the polymorphic HLA loci are most commonly typed by serological methods. Such typing is used in paternity determinations, transplant and transfusion histocompatibility testing, blood component therapy, anthropological studies and in disease association correlation to diagnose diseases or predict susceptibility to diseases. The major drawbacks to such HLA typing, particularly of the Class II loci, are the complexity of the sera and the lack of widespread availability of standard sera necessary to conduct the tests. In addition, since serological typing is based on reactions of sera with the HLA gene products, it is not useful for HLA typing for HLA-deficient individuals in whom HLA products are reduced or absent.
Serological or cellular HLA typing techniques require the presence of detectable levels of HLA proteins on the surface of lymphocytes. In some cases (e.g., HLA-deficient SCID or cellular depletion due to chemotherapy), the levels of the HLA proteins or number of available cells are inadequate to achieve reliable HLA typing. Another limitation of traditional typing methods is the inability to resolve all functionally important HLA alleles. These circumstances have prompted the development of methods for analysis of HLA polymorphism at the genetic level, as described in Bidwell, J., 1988, Immunology Today 9:18-23, and Angelini et al., 1986, Proc. Natl. Acad. Sci. USA, 83:4489-4493.
Non-serological HLA typing methods have been proposed to overcome drawbacks with serological typing. One such method involves the use of DNA restriction fragment length polymorphism (RFLP) as a basis for HLA typing. See Erlich U.S. Pat. No. 4,582,788, issued Apr. 15, 1986. Polymorphism detected by this method is located in both coding and noncoding sequences of the genome. Therefore, RFLP often does not directly measure functional polymorphism, but relies upon linkage disequilibrium between polymorphism in non-coding regions and the coding region. RFLP analysis has been used for typing an HLA-deficient severe combined immunodeficiency (SCID) patient, but its utility as a routine method is limited by laborious procedures, inadequate resolution of alleles, and difficulty in interpreting data for certain combinations of alleles.
Some RFLP and similar typing methods utilize labelled obligonucleotides to identify specific HLA and DNA sequences. In particular, the use of oligonucleotide probes have been found advantageous in HLA-DR typing in identifying variant genes encoding products which are not detectable serologically. See Angelini et al., above, Scharf et al., Science, Vol. 233, No. 4768, pp. 1076-1078, Cox et al., Am. J. Hum. Gen., 43:954-963, 1988, Tiercy et al., Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 198-202, 1988, and Tiercy et al., Hum. Immunol. 24, pp. 1-14 (1989). Sequence-specific oligonucleotide probe hybridization (SSOPH) can discriminate single base pair mismatches, which is equivalent to detecting a single amino acid polymorphism in HLA proteins.
The polymerase chain reaction (PCR) process, as described in Mullis U.S. Pat. No. 4,683,202, issued Jul. 28, 1987, allows the amplification of genomic DNA and has given rise to more convenient HLA typing procedures. HLA-DQ alpha and HLA-DP alpha and beta genes have been amplified, and then sequenced or hybridized with oligonucleotide probes. See Saiki et al., Nature, Vol. 324, pp. 163-166, 1986, Bugawan et al., J. Immunol., Vol. 141, No. 12, pp. 4024-4030, 1988, and Gyllensten et al., Proc. Natl. Acad. Sci. USA, Vol. 85, pp. 7652-7656, 1988. However, these methods have limited reliability due to the tendency of the probes to bind with greater or lesser specificity depending on the reaction conditions employed. The present invention addresses the latter problem and provides certain specific probes and primers useful in HLA-DR typing.
The invention further concerns the use of HLA typing methods for tissue matching, especially for purposes of tissue transplantation. Bone marrow transplants, in particular, are the treatment of choice for certain immunodeficiency syndromes and hematological disorders. The success of bone marrow transplantation is influenced by the degree of HLA compatibility between recipient and donor. However, aberrant HLA expression sometimes makes it difficult, if not impossible, to determine the patient's HLA type by standard serological and cellular techniques.
Successful bone marrow transplantation depends on the degree of HLA matching between donor/recipient pairs. This results from the physiological role of human lymphocyte antigens in self-restriction of cellular interactions during and immune response. See Schwartz, Ann. Rev. Immunol. 3:27-261, 1985. If polymorphic residues in the HLA proteins are mismatched, the immune system may recognize the cells bearing the mismatched HLA as foreign. The consequences of such mismatching include graft-versus-host disease (GVHD), graft rejection, and failure to reconstitute a competent immune system. See Hows, Bone Marrow Transplantation, 1:259-264. These problems are minimized by selection of HLA-matched siblings as donors. Unfortunately, this option is available for only about 30-40% of patients who could benefit from a bone marrow transplant. In the remaining patients (60-70%), HLA typing with high resolving power is necessary for selection of an optimally matched, unrelated donor.
More than 40 variant HLA-DR beta alleles have been identified among the population, and more are being identified on an ongoing basis. It has been suggested that, within the population, HLAs may exist as numerous combinations of a group of polymorphic sequences, rather than a limited number of discrete alleles. The present invention provides a comprehensive process for HLA typing which overcomes many problems associated with serological typing methods, for example, the requirement for HLA-expression, viable cells, and limitations in resolving certain alleles. The invention further provides a method of tissue matching which has provided superior results in the context of bone marrow transplantation.